Thermodynamic and kinetic analyses of androgen and glucocorticoid receptor-promoter interactions suggest phylogenetic partitioning of energetics and the presence of functionally distinct DNA-bound state
Year: 2015
Type: dissertation
Abstract: The fundamental principles governing transcriptional activation in higher eukaryotes are not well understood. As a step towards defining these principles, we seek a quantitative understanding of transcription factor interactions. To this end, we use thermodynamics coupled with statistical thermodynamic modeling and transient state kinetics to elucidate mechanisms of transcription factor interactions. As a model system, our lab studies the steroid receptor family of ligand-activated transcription factors. This family is comprised of two subgroups. Subgroup 3A consists of the two estrogen receptor isoforms (ER-α and ER-β), receptors that are closely related to an ancient ER-like steroid receptor ancestor. Subgroup 3C consists of more distantly related proteins: androgen receptor (AR), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and the two progesterone receptor isoforms (PR-A and PR-B). Here, we investigated the self-assembly and promoter energetics of wild type AR and a point mutation associated with prostate cancer progression, T877A. I first found that the AR constructs show no evidence of dimerization. Although this is in contrast to the literature, it is in-line with our previous studies on the other subgroup 3C members. I also found that the AR constructs display strong intersite cooperativity at a two-site promoter. Again, this is consistent with our studies on PR-B and GR, two other subgroup 3C members. Taken together, these results are in contrast to the more distantly related receptor ER-α. Thus, the steroid receptors partition their self-association and promoter assembly energetics in-line with their phylogenetic divergence. In addition to thermodynamic studies, the dissociation kinetics of GR from a panel of six response elements and the mouse mammary tumor virus promoter were examined. In contrast to the literature, GR dissociates from its response elements on the timescale of seconds to tens of seconds, indicating that receptor-DNA interactions are primarily responsible for transient behavior. Also, GR dissociation is biphasic in character, suggesting receptor interactions with DNA are more complex than a rigid body interaction. Direct fitting of kinetic data indicates that GR dissociation involves an isomerization between two distinct GR-DNA states, suggesting two functionally distinct states.
DOI: 10.25677/js6d-ka97
Author: De Angelis, Rolando Wray
Language: English
Subfield: Endocrinology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2279655414